Enzymes constitute a family of proteins involved in catalysing chemical reactions within living organisms. As a result of their importance, there are numerous situations in which it is necessary and/or beneficial to measure enzyme levels, and importantly, enzyme activity.
In particular, increases in enzyme activity have been found to correlate with specific conditions and/or diseases. For example up-regulated protease activity has been associated with many aspects of cancer progression. The measurement of enzyme activity in samples taken from individuals with a particular condition or suspected of having a specific condition or disease may therefore be useful for prognostic or diagnostic purposes.
Within the enzyme family, there are many classes of enzyme that act by facilitating substrate cleavage. For example, peptidases and proteases catalyse the hydrolysis of peptide bonds within their respective substrates. In the past, researchers have, in some cases, sought to measure this type of activity using kits or devices that measure release of a fragment or ‘leaving group’ from the initial enzyme substrate.
Assays based on this fundamental principle have been refined such that in some cases, inventors have described engineered substrate molecules linked to reporter moieties. Cleavage of the substrate by the enzyme to be detected, if present, leads to release of said reporter, which can be detected by a range of techniques available to those skilled in the art. An assay of this type is described for example in US2006/0003394.
Others have sought to develop assays for the measurement of enzyme activity based around the principle of discriminating between modified and unmodified forms of an enzyme substrate. In this regard, WO2009/024805 describes an enzyme detection device utilising a “substrate recognition molecule” (SRM) carrying a detectable label, wherein the SRM specifically binds to the enzyme substrate in either the unmodified or modified state.
Problems associated with the assays described to date include, in particular, the accuracy that can be achieved using the formats described. In particular, US2006/0003394 describes a kit wherein the enzyme-substrate mixture is applied to a chromatographic medium. Immobilisation of the substrate-reporter “reactive complex” at an upstream site occurs in the absence of enzyme whereas, in the presence of enzyme, cleavage of the reactive complex results in flow of the reporter to a downstream site. Since the enzyme, if present, can continue to cleave reactive complex immobilised at the upstream site, any signal generated by the presence of reporter at either site is subject to change with time, such that there is no clear endpoint to the reaction. This can create significant problems with the accuracy and reproducibility of data generated using this assay format.